Oxidoreductases for the stereoselective reduction of keto compounds

ABSTRACT

The invention relates to a process for the enantioselective enzymatic reduction of a keto compound to the corresponding chiral hydroxy compound, wherein the keto compound is reduced with an oxidoreductase in the presence of a cofactor, and is characterized in that an oxidoreductase is used which has an amino acid sequence in which (a) at least 70% of the amino acids are identical to the amino acids of one of the amino acid sequences SEQ ID No 1, SEQ ID No 6 and SEQ ID No 8, or (b) at least 55% of the amino acids are identical to the amino acids of the amino acid sequence SEQ ID No 2, or (c) at least 65% of the amino acids are identical to the amino acids of the amino acid sequence SEQ ID No 3, or (d) at least 75% of the amino acids are identical to the amino acids of the amino acid sequence SEQ ID No 4, or (e) at least 65% of the amino acids are identical to the amino acids of the amino acid sequence SEQ ID No 5, or (f) at least 50% of the amino acids are identical to the amino acids of the amino acid sequence SEQ ID No 7, or (g) at least 72% of the amino acids are identical to the amino acids of the amino acid sequence SEQ ID No 129.

The present invention relates to a process for the enantioselectiveenzymatic reduction of a keto compound to the corresponding chiralhydroxy compound, wherein the keto compound is reduced with anoxidoreductase. Furthermore, the invention relates to newoxidoreductases for use in the enantioselective reduction of ketocompounds to chiral hydroxy compounds.

Optically active hydroxy compounds are valuable chirons with broadapplicability for the synthesis of pharmacologically active compounds,aromatic substances, pheromones, agricultural chemicals and enzymeinhibitors. Thereby, an increasing demand for chiral compounds and thuschiral synthesis technologies can be noted particularly in thepharmaceutical industry, since, in the future, racemic compounds willhardly be used as pharmaceutical preparations.

The asymmetric reduction of prochiral keto compounds is a sector ofstereoselective catalysis, wherein biocatalysis constitutes a powerfulcompetitive technology versus chemical catalysis. The chemicalasymmetric hydrogenation requires the use of highly toxic andenvironmentally harmful heavy metal catalysts, of extreme and thusenergy-intensive reaction conditions as well as large amounts of organicsolvents. Furthermore, these methods are often characterized by sidereactions and insufficient enantiomeric excesses.

In nature, reductions of prochiral keto compounds to hydroxy compoundsand vice versa occur in numerous biochemical pathways, both in theprimary metabolism and in the secondary metabolism, in every organismand are catalyzed by different types of secondary alcohol dehydrogenasesand oxidoreductases. Normally, these enzymes are cofactor-dependent.

The basic feasibility of using biocatalysts for the reduction ofprochiral keto compounds to chiral hydroxy compounds was repeatedlydemonstrated in the past on the basis of model systems, wherein bothisolated oxidoreductases and various whole-cell biotransformationsystems were used for the task. Thereby, the biocatalytic approachturned out to be advantageous essentially with regard to mild reactionconditions, lack of byproducts and often significantly better achievableenantiomeric excesses. The use of isolated enzymes is advantageous overmethods involving whole cells with regard to the achievable enantiomericexcess, the formation of degradation products and byproducts as well aswith regard to the product isolation. Moreover, the use of whole-cellprocesses is not possible for every chemical company, since specificequipment and know-how is required therefor.

Recently, it has been possible to demonstrate that the use of isolatedoxidoreductases in aqueous/organic two-phase systems with organicsolvents is extremely efficient, economical and feasible also at highconcentrations (>5%). In the described systems, the keto compound to bereduced, which usually is poorly soluble in water, thereby forms theorganic phase together with the organic solvent. Also, the organicsolvent itself can partly be dispensed with. In that case, the organicphase is formed from the keto compound to be reduced (DE 10119274, DE10327454.4, DE 103 37 401.9, DE 103 00335.5). Coenzyme regeneration isthereby achieved by the concurrent oxidation of secondary alcohols, forwhich, in most cases, the inexpensive water-miscible 2-propanol is used.

Examples of Suitable R- and S-Specific Oxidoreductases andDehydrogenases of High Enantioselectivity are:

Carbonyl reductase from Candida parapsilosis (CPCR) (U.S. Pat. No.5,523,223 and U.S. Pat. No. 5,763,236, (Enzyme Microb Technol. 1993November; 15(11):950-8)) and Pichia capsulata (DET0327454.4). Carbonylreductase from Rhodococcus erythropolis (RECR) (U.S. Pat. No.5,523,223), Norcardia fusca (Biosci. Biotechnol. Biochem., 63 (10)(1999), pp. 1721-1729), (Appl Microbiol Biotechnol. 2003 September;62(4):380-6. Epub 2003 Apr. 26), and Rhodococcus ruber (J Org. Chem.2003 Jan. 24; 68(2):402-6.).

R-specific secondary alcohol dehydrogenases from organisms of the genusLactobacillus (Lactobacillus kefir (U.S. Pat. No. 5,200,335),Lactobacillus brevis (DE 19610984 A1) (Acta Crystallogr D BiolCrystallogr. 2000 December; 56 Pt 12:1696-8), Lactobacillus minor(DE10119274) or Pseudomonas (U.S. Pat. No. 05,385,833)(Appl MicrobiolBiotechnol. 2002 August; 59(4-5):483-7. Epub 2002 Jun. 26, J. Org. Chem.1992, 57, 1532)

However, the enzymes known today are not nearly sufficient forexploiting the entire market potential of stereoselective reductions ofketo compounds. On the one hand, this can be explained by the fact thatthe individual enzymes have very different properties with respect tosubstrate spectrum, pH optima as well as temperature and solventstabilities, which often supplement each other. Therefore, evenrelatively similar homologous enzymes may exhibit a completely differentconversion behaviour with regard to one particular substrate. On theother hand, not nearly all of the enzymes described are cloned andoverexpressible to a sufficient extent, which means that these enzymesare not available for industrial use. For exploiting the syntheticpotential of the enzymatic asymmetric hydrogenation as extensively aspossible, it is therefore necessary to be in possession of a portfolioof different industrially accessible oxidoreductases which is as broadas possible, which oxidoreductases are furthermore suitable for use inaqueous/organic two-phase systems with organic solvents.

The subject matter of the present invention is now a number of novel,enantioselective R- and S-specific oxidoreductases characterized by goodstability in aqueous/organic two-phase systems as well as by goodexpressibility in Escherichia coli (>500 units/g E. coli wet biomass),as well as a process for the enantioselective enzymatic reduction of aketo compound to the corresponding chiral hydroxy compound.

The oxidoreductases according to the invention are characterized in thatthey have an amino acid sequence in which

(a) at least 70% of the amino acids are identical to the amino acids ofone of the amino acid sequences SEQ ID No 1, SEQ ID No 6 and SEQ ID No8, or(b) at least 55% of the amino acids are identical to the amino acids ofthe amino acid sequence SEQ ID No 2, or(c) at least 65% of the amino acids are identical to the amino acids ofthe amino acid sequence SEQ ID No 3, or(d) at least 75% of the amino acids are identical to the amino acids ofthe amino acid sequence SEQ ID No 4, or(e) at least 65% of the amino acids are identical to the amino acids ofthe amino acid sequence SEQ ID No 5, or(f) at least 50% of the amino acids are identical to the amino acids ofthe amino acid sequence SEQ ID No 7.(g) at least 72% of the amino acids are identical to the amino acids ofthe amino acid sequence SEQ ID No 129.

The polypeptide according to SEQ ID No 1 can be obtained from yeasts, inparticular from yeasts of the genus Rhodotorula, in particular fromRhodotorula mucilaginosa. A further subject matter of the invention is anucleic acid sequence SEQ ID No 9, which codes for the protein havingthe amino acid sequence SEQ ID No 1.

The oxidoreductase from Rhodotorula mucilaginosa reduces, for example,2-octanone to S-2-octanol and preferably oxidizes S-2-octanol out of thetwo enantiomers of the 2-octanol. The oxidoreductase from Rhodotorulamucilaginosa is, for example, a homodimer having a molecular weightdetermined in the SDS—gel of 30±2 k Da. The pH optimum for the reductionreaction ranges from 7.0 to 8.0 for said oxidoreductase, and the pHoptimum for the oxidation reaction is in the range of from 8.5-10. Theoxidoreductase from Rhodotorula mucilaginosa exhibits good temperatureand pH stabilities and shows only minor activity losses in the pH rangeof from 5.5 to 10 and at temperatures of up to 35° C., even withincubation periods of several hours. Furthermore, the oxidoreductasefrom Rhodotorula mucilaginosa exhibits high stability in organicsolvents.

Polypeptides according to SEQ ID No 2 or SEQ ID No 8 can be obtainedfrom yeasts, in particular from yeasts of the genera Pichia, Candida,Pachysolen, Debaromyces or Issatschenkia, in particular from Pichiafarinosa DSMZ 3316 or Candida nemodendra DSMZ 70647. A further subjectmatter of the invention is a nucleic acid sequence SEQ ID No 10 and anucleic acid sequence SEQ ID No 16, which code for the amino acidsequences SEQ ID No 2 and SEQ ID No 8, respectively. The oxidoreductasepreferably reduces 2-butanone to R-2-butanol and preferably oxidizesR-2-butanol out of the two enantiomers of the 2-butanol.

The oxidoreductase from Pichia farinosa exhibits a significantly higheractivity towards R-2-butanol and 2-propanol than towards R-2-octanol, inaddition, the enzyme exhibits a significantly higher activity towardsacetone and 2-butanone than towards 2-octanone.

However, the oxidoreductase from Candida nemodendra exhibits a similaractivity towards R-2-butanol, 2-propanol and R-2-octanol, in addition,the enzyme also exhibits an approximately similar activity towards2-octanone.

The oxidoreductase from Pichia farinosa is a homodimer having amolecular weight determined in the SDS—gel of 27±2 k Da. The pH optimumfor the reduction reaction ranges from 5.0 to 6.0 for saidoxidoreductase, and the pH optimum for the oxidation reaction rangesfrom 7.5-10. The oxidoreductase from Pichia farinosa exhibits good pHand solvent stabilities and shows only minor activity losses in the pHrange of from 5.5 to 10, even with incubation periods of several hours.

The oxidoreductase from Candida nemodendra is a homomer having amolecular weight determined in the SDS—gel of 27±2 k Da. The pH optimumfor the reduction reaction is at pH 6 for said oxidoreductase, and thepH optimum for the oxidation reaction ranges from 10-11. Theoxidoreductase from Candida nemodendra exhibits good pH and solventstabilities and shows only minor activity losses in the pH range of from6.5 to 9.5, even with incubation periods of several hours.

The polypeptides according to SEQ ID No 3 or SEQ ID No 7 can be obtainedfrom yeasts, in particular from yeasts of the genera Pichia and Candida,in particular from Pichia stipidis DSMZ 3651 and Pichia trehalophilaDSMZ 70391. A further subject matter of the invention is a nucleic acidsequence SEQ ID No 11 and a nucleic acid sequence SEQ ID No 15, whichencode polypeptides SEQ ID No 3 and SEQ ID No 7, respectively.

The carbonyl reductases from yeasts of the genera Pichia and Candida,which have at least 65% identity to the amino acid sequence SEQ ID No 3or at least 50% identity to the amino acid sequence SEQ ID No 7,preferably reduce 2-octanone to S-2-octanol and preferably oxidizeS-2-octanol out of the two enantiomers of the 2-octanol. They are alsoparticularly suitable for the reduction of 4-haloacetoacetate esters toR-4-halo-3-hydroxybutyric acid esters.

The oxidoreductase from Pichia stipidis is a homodimer having amolecular weight determined in the SDS—gel of 36±2 k Da. The pH optimumfor the reduction reaction ranges from 5.5 to 6.5 for saidoxidoreductase, and the pH optimum for the oxidation reaction rangesfrom 6.5-8.0. The oxidoreductase from Pichia stipidis exhibits good pHand solvent stabilities and shows only minor activity losses in the pHrange of from 5.5 to 10, even with incubation periods of several hours.

The oxidoreductase from Pichia trehalophila is a homomer having amolecular weight determined in the SDS—gel of 36±2 k Da. The pH optimumfor the reduction reaction ranges from 7-7.5 for said oxidoreductase,and the pH optimum for the oxidation reaction ranges from 7-8.

The polypeptide according to SEQ ID No 4 can be obtained from bacteriaof the class Leuconostoc, in particular from Leuconostoc carnosum DSMZ5576. A further subject matter of the invention is a nucleic acidsequence SEQ ID No 12, which codes for a protein having the amino acidsequence SEQ ID No 4. The polypeptide is particularly suitable for thereduction of 2-octanone to R-2-octanol and for the oxidation ofR-2-octanol. It is also very suitable for the reduction of4-haloacetoacetate esters to S-4-halo-3-hydroxybutyric acid esters.

The oxidoreductase from Leuconostoc camosum is a homodimer having amolecular weight determined in the SDS—gel of 27±2 k Da. The pH optimumfor the reduction reaction ranges from 5.0 to 6.0 for saidoxidoreductase, and the pH optimum for the oxidation reaction rangesfrom 6.0-9.0. The oxidoreductase from Leuconostoc camosum exhibits goodtemperature, pH and solvent stabilities and shows only minor activitylosses in the pH range of from 4.5 to 10 and at temperatures of up to35° C., even with incubation periods of several hours.

The polypeptide according to SEQ ID No 5 can be obtained from bacteriaof the class Actinobacteria, in particular from bacteria of the classMicrobacterium, in particular from Microbacterium spec. DSMZ 20028. Afurther subject matter of the invention is a nucleic acid sequence SEQID No 13, which codes for the protein having the amino acid sequence SEQID No 5. The polypeptide is very suitable for the reduction of2-octanone to S-2-octanol and preferably oxidizes S-2-octanol out of thetwo enantiomers of the 2-octanol. It is also very suitable for thereduction of 4-haloacetoacetate esters to R-4-halo-3-hydroxybutyric acidesters.

The oxidoreductase from Microbacterium spec. DSMZ 20028 is, for example,a homotetramer having a molecular weight determined in the SDS—gel of35±2 k Da. The pH optimum for the reduction reaction ranges from 6.0 to7.5 for said oxidoreductase, and the pH optimum for the oxidationreaction ranges from 7.5-9.5. The oxidoreductase from Microbacteriumspec exhibits good temperature, pH and solvent stabilities and showsonly minor activity losses in the pH range of from 4.5 to 10 and attemperatures of up to 50° C., even with incubation periods of severalhours.

The polypeptide according to SEQ ID No 6 can be obtained from bacteriaof the class Actinobacteria, in particular from bacteria of the classGordonia, in particular from Gordonia rubripertincta DSMZ 43570. Afurther subject matter of the invention is a nucleic acid sequence SEQID No 14, which codes for the protein having the amino acid sequence SEQID No 6. The polypeptide is very suitable for the reduction of2-octanone to S-2-octanol and preferably oxidizes S-2-octanol out of thetwo enantiomers of the 2-octanol. It is also very suitable for thereduction of 4-haloacetoacetate esters to R-4-halo-3-hydroxybutyric acidesters.

The oxidoreductase from Gordonia rubripertincta DSMZ 43570 is a homomerhaving a molecular weight determined in the SDS—gel of 41±3 k Da. The pHoptimum for the reduction reaction ranges from 4.5 to 5.5 for saidoxidoreductase, and the pH optimum for the oxidation reaction rangesfrom 7.5 to 9.5. The oxidoreductase from Gordonia rubripertincta DSMZ43570 exhibits good temperature, pH and solvent stabilities and showsonly minor activity losses in the pH range of from 4.5-10 and attemperatures of up to 55° C., even with incubation periods of severalhours.

The polypeptide according to SEQ ID No 129 can be obtained from yeasts,in particular from yeasts of the genera Lodderomyces, in particular fromLodderomyces elongisporus DSMZ 70320. A further subject matter of theinvention is a nucleic acid sequence SEQ ID No 130, which codes for theprotein having the amino acid sequence SEQ ID No 129. The polypeptide isvery suitable for the reduction of 2-octanone to S-2-octanol andpreferably oxidizes S-2-octanol out of the two enantiomers of the2-octanol. It is also very suitable for the reduction of4-haloacetoacetate esters to R-4-halo-3-hydroxybutyric acid esters.

Furthermore, the invention relates to fusion proteins which arecharacterized in that they represent oxidoreductases having the aminoacid sequences SEQ ID No 1, SEQ ID No 2, SEQ ID No 3, SEQ ID No 4, SEQID No 5, SEQ ID No 6, SEQ ID No 7, SEQ ID No 8, SEQ ID No 129 orhomologues thereof, which are peptidically linked to a furtherpolypeptide at the N-terminal or carboxy-terminal end. Fusion proteinscan, for example, be separated more easily from other proteins or can berecombinantly expressed in larger amounts.

Furthermore, the invention relates to antibodies which specifically bindto oxidoreductases according to SEQ ID No 1, SEQ ID No 2, SEQ ID No 3,SEQ ID No 4, SEQ ID No 5, SEQ ID No 6, SEQ ID No 7, SEQ ID No 8, SEQ IDNo 129 or to homologues thereof. The production of these antibodies isperformed according to known methods by immunization of appropriatemammals and subsequent recovery of the antibodies. The antibodies can bemonoclonal or polyclonal.

Comparisons of amino acid sequences can, for example, be conducted inthe internet in protein databases such as, e.g., SWISS-PROT, PIR as wellas in DNA databases such as, e.g., EMBL, GenBank etc., using theFASTA-program or the BLAST-program.

In doing so, the optimal alignment is determined by means of the BLASTalgorithm (Basic Local Alignement Search Tool) (Altschul et al. 1990,Proc. Natl. Acd. Sci. USA. 87: 2264-2268). As a basis, the PAM30 matrixis used as a scoring matrix for evaluating the sequence similarity.(Dayhoff; M. O., Schwarz R. M., Orcutt B. C. 1978. “A model ofevolutionary change in Proteins” in “Atlas of Protein Sequence andstructure” 5(3) M. O. Dayhoff (ed) 345-352, National Biomedical Researchfoundation).

Furthermore, the invention relates to protein fragments which arecharacterized in that they represent fragments of the amino acidsequence SEQ ID No 1, with a number of more than 26 amino acids perfragment.

A further subject matter of the invention is a microbial carbonyldehydrogenase which comprises the amino acid sequence MPATLRLDK (SEQ IDNo 17) N-terminally and/or the amino acid sequence QALAAPSNLAPKA (SEQ IDNo 18) C-terminally and/or one of the internal partial sequencesVEIIKTQVQD (SEQ ID No 19), KVAIITGGASGIGL (SEQ ID No 20), SCYVTPEG (SEQID No 21), TDFKVDGG (SEQ ID No 22), VMFNNAGIMH (SEQ ID No 23) orVHAREGIRIN (SEQ ID No 24).

Furthermore, the invention relates to protein fragments which arecharacterized in that they represent fragments of the amino acidsequence SEQ ID No 2, with a number of more than 15 amino acids perfragment.

A further subject matter of the invention is a microbial carbonyldehydrogenase which comprises the amino acid sequence MAYNFTNKVA (SEQ IDNo 25) N-terminally and/or the amino acid sequence TTLLVDGGYTAQ (SEQ IDNo 26) C-terminally and/or one of the internal partial sequencesEYKEAAFTN (SEQ ID No 27), NKVAIITGGISGIGLA (SEQ ID No 28), DVNLNGVFS(SEQ ID No 29), HYCASKGGV (SEQ ID No 30), NCINPGYI (SEQ ID No 31) orLHPMGRLGE (SEQ ID No 32).

Furthermore, the invention relates to protein fragments which arecharacterized in that they represent fragments of the amino acidsequence SEQ ID No 3, with a number of more than 15 amino acids perfragment.

A further subject matter of the invention is a microbial carbonyldehydrogenase which comprises the amino acid sequence MSIPATQYGFV (SEQID No 33) N-terminally and/or the amino acid sequence SAYEGRVVFKP (SEQID No 34) C-terminally and/or one of the internal partial sequencesCHSDLHAIY (SEQ ID No 35), GYQQYLLVE (SEQ ID No 36), TFDTCQKYV (SEQ ID No37), LLTPYHAM (SEQ ID No 38), LVSKGKVKP (SEQ ID No 39), GAGGLGVNG (SEQID No 40), IQIAKAFGAT (SEQ ID No 41) or LGSFWGTS (SEQ ID No 42).

Furthermore, the invention relates to protein fragments which arecharacterized in that they represent fragments of the amino acidsequence SEQ ID No 4, with a number of more than 18 amino acids perfragment.

A further subject matter of the invention is a microbial carbonyldehydrogenase which comprises the amino acid sequence MTDRLKNKVA (SEQ IDNo 43) N-terminally and/or the amino acid sequence AEFVVDGGYLAQ (SEQ IDNo 44) C-terminally and/or one of the internal partial sequencesVVITGRRAN (SEQ ID No 45), GGASIINMS (SEQ ID No 46), TQTPMGHI (SEQ ID No47) or GYIKTPLVDG (SEQ ID No 48).

Furthermore, the invention relates to protein fragments which arecharacterized in that they represent fragments of the amino acidsequence SEQ ID No 5, with a number of more than 18 amino acids perfragment.

A further subject matter of the invention is a microbial carbonyldehydrogenase which comprises the amino acid sequence MKALQYTKIGS (SEQID No 49) N-terminally and/or the amino acid sequence LAAGTVRGRAVIVP(SEQ ID No 50) C-terminally and/or one of the internal partial sequencesCHSDEFVMSLSE (SEQ ID No 51), VYGPWGCGRC (SEQ ID No 52), VSLTDAGLTPYHA(SEQ ID No 53), LRAVSAATVIAL (SEQ ID No 54) or DFVGADPTI (SEQ ID No 55).

Likewise, the invention relates to protein fragments which arecharacterized in that they represent fragments of the amino acidsequence SEQ ID No 6, with a number of more than 26 amino acids perfragment.

A further subject matter of the invention is a microbial carbonyldehydrogenase which comprises the amino acid sequence MKAIQIIQ (SEQ IDNo 56) N-terminally and/or the amino acid sequence DLRGRAVVVP (SEQ ID No57) C-terminally and/or one of the internal partial sequences TAAGACHSD(SEQ ID No 58), TPYHAIKPSLP(SEQ ID No 59), DFVGLQPT (SEQ ID No 60),VYGAWGCG (SEQ ID No 61), DDARHLVP (SEQ ID No 62), MTLGHEGA (SEQ ID No63) or GGLGHVGIQLLRHL (SEQ ID No 64).

Furthermore, the invention relates to a cloning vector comprising one orseveral nucleic acid sequences coding for the carbonyl reductasesaccording to SEQ ID No 1, SEQ ID No 2, SEQ ID No 3, SEQ ID No 4, SEQ IDNo 5, SEQ ID No 6, SEQ ID No 7, SEQ ID No 8, SEQ ID No 129 or homologuesthereof. Moreover, the invention comprises a cloning vector which, inaddition to the carbonyl reductase, includes a suitable enzyme for theregeneration of NAD(P) such as, e.g., formate dehydrogenases, alcoholdehydrogenases or glucose dehydrogenase.

Furthermore, the invention relates to an expression vector located in abacterial, insect, plant or mammalian cell and comprising a nucleic acidsequence which codes for the carbonyl reductases according to SEQ ID No1, SEQ ID No 2, SEQ ID No 3, SEQ ID No 4, SEQ ID No 5, SEQ ID No 6, SEQID No 7, SEQ ID No 8, SEQ ID No 129 or homologues thereof and is linkedin an appropriate way to an expression control sequence. Furthermore,the invention relates to a recombinant host cell which is a bacterial,yeast, insect, plant or mammalian cell and has been transformed ortransfected with such an expression vector as well as to a productionprocess for obtaining a carbonyl reductase based on the cultivation ofsuch a recombinant host cell.

Suitable cloning vectors are, for example, ppCR-Script, pCMV-Script,pBluescript (Stratagene), pDrive cloning Vector (Quiagen, Hilden,Germany), pS Blue, pET Blue, pET LIC-vectors (Novagen, Madison, USA) andTA-PCR cloning vectors (Invitrogen, Karlsruhe, Germany).

Suitable expression vectors are, for example, pKK223-3, pTrc99a, pUC,pTZ, pSK, pBluescript, pGEM, pQE, pET, PHUB, pPLc, pKC30, pRM1/pRM9,pTrxFus, pAS1, pGEx, pMAL or pTrx.

Suitable expression control sequences are, for example, trp-lac(tac)-promoter, trp-lac (trc)-promoter, lac-promoter, T7-promoter orλpL-promoter.

The oxidoreductases according to SEQ ID No 1, SEQ ID No 2, SEQ ID No 3,SEQ ID No 4, SEQ ID No 5, SEQ ID No 6, SEQ ID No 7, SEQ ID No 8, SEQ IDNo 129 or homologues thereof can be obtained in such a manner that theabove-mentioned recombinant E. coli cells are cultivated, the expressionof the respective oxidoreductase is induced and subsequently, afterabout 10 to 18 hours (h), the cells are digested by ultrasonictreatment, by wet grinding with glass beads in a globe mill (Retsch,GmbH, Haan Germany 10 min, 24 Hz) or using a high-pressure homogenizer.The cell extract obtained can either be used directly or purifiedfurther. For this purpose, the cell extract is, e.g., centrifuged andthe supernatant obtained is subjected to ion exchange chromatography,for example, by ion exchange chromatography on Q-Sepharose Fast Flow®(Pharmacia).

Furthermore, the invention relates to a process for the enantioselectiveenzymatic reduction of a keto compound to the corresponding chiralhydroxy compound, wherein the keto compound is reduced with anoxidoreductase in the presence of a cofactor, characterized in that anoxidoreductase is used which has an amino acid sequence in which

(a) at least 70% of the amino acids are identical to the amino acids ofone of the amino acid sequences SEQ ID No 1, SEQ ID No 6 and SEQ ID No8, or(b) at least 55% of the amino acids are identical to the amino acids ofthe amino acid sequence SEQ ID No 2, or(c) at least 65% of the amino acids are identical to the amino acids ofthe amino acid sequence SEQ ID No 3, or(d) at least 75% of the amino acids are identical to the amino acids ofthe amino acid sequence SEQ ID No 4, or(e) at least 65% of the amino acids are identical to the amino acids ofthe amino acid sequence SEQ ID No 5, or(f) at least 50% of the amino acids are identical to the amino acids ofthe amino acid sequence SEQ ID No 7.(g) at least 72% of the amino acids are identical to the amino acids ofthe amino acid sequence SEQ ID No 129.

A further preferred embodiment of the process according to the inventionconsists in that the keto compound has the general formula I

R₁—C(O)—R₂  (I)

wherein R₁ stands for one of the moieties1) —(C₁-C₂₀)-alkyl, wherein alkyl is linear-chain or branched,2) —(C₂-C₂₀)-alkenyl, wherein alkenyl is linear-chain or branched andoptionally contains up to four double bonds,3) —(C₂-C₂₀)-alkynyl, wherein alkynyl is linear-chain or branched andoptionally contains up to four triple bonds,4) —(C₆-C₁₄)-aryl,5) —(C₁-C₈)-alkyl-(C₆-C₁₄)-aryl,6) —(C₅-C₁₄)-heterocycle which is unsubstituted or substituted one, twoor three times by —OH, halogen, —NO₂ and/or —NH₂, or7) —(C₃-C₇)-cycloalkyl,wherein the moieties mentioned above under 1) to 7) are unsubstituted orsubstituted one, two or three times, independently of each other, by—OH, halogen, —NO₂ and/or —NH₂,and R₂ stands for one of the moieties8) —(C₁-C₆)-alkyl, wherein alkyl is linear-chain or branched,9) —(C₂-C₆)-alkenyl, wherein alkenyl is linear-chain or branched andoptionally contains up to three double bonds,10) —(C₂-C₆)-alkynyl, wherein alkynyl is linear-chain or branched andoptionally contains two triple bonds, or11) —(C₁-C₁₀)-alkyl-C(O)—O—(C₁-C₆)-alkyl, wherein alkyl is linear orbranched and is unsubstituted or substituted one, two or three times by—OH, halogen, —NO₂ and/or —NH₂, wherein the moieties mentioned aboveunder 8) to 11) are unsubstituted or substituted one, two or threetimes, independently of each other, by —OH, halogen, —NO₂ and/or —NH₂.

Furthermore, the invention relates to a process for the enantioselectiveenzymatic reduction of a keto compound to the corresponding chiralhydroxy compound, wherein the keto compound is reduced with anoxidoreductase in the presence of a cofactor, which process ischaracterized in that an oxidoreductase is used which

-   (a) is encoded by a nucleic acid sequence from the group of SEQ ID    No 9, SEQ ID No 10, SEQ ID No 11, SEQ ID No 12, SEQ ID No 13 and SEQ    ID No 14, SEQ ID No 15, SEQ ID No 16 and SEQ ID No 130, or which-   (b) is encoded by a nucleic acid sequence the complementary strand    of which hybridizes with one of the nucleic acid sequences mentioned    in (a) under highly stringent conditions.

By the term “1”, aromatic carbon moieties comprising 6 to 14 carbonatoms within the ring are understood —(C₆-C₁₄)-aryl moieties are, forinstance, phenyl, naphthyl, e.g., 1-naphthyl, 2-naphthyl, biphenylyl,e.g., 2-biphenylyl, 3-biphenylyl and 4-biphenylyl, anthryl or fluorenyl.Biphenylyl moieties, naphthyl moieties and in particular phenyl moietiesare preferred aryl moieties. By the term “halogen”, an element from thefamily of fluorine, chlorine, bromine or iodine is understood. By theterm “—(C₁-C₂₀)-alkyl”, a hydrocarbon moiety is understood the carbonchain of which is linear-chain or branched and comprises 1 to 20 carbonatoms, for example, methyl, ethyl, propyl, isopropyl, butyl, tertiarybutyl, pentyl, hexyl, heptyl, octyl, nonenyl or decanyl. By the term“—C₀-alkyl”, a covalent bond is understood.

By the term “—(C₃-C₇)-cycloalkyl”, cyclic hydrocarbon moieties such ascyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl areunderstood.

The term “—(C₅-C₁₄)-heterocycle” stands for a monocyclic or bicyclic5-membered to 14-membered heterocyclic ring which is partially orcompletely saturated. N, O and S are examples of heteroatoms. Moietiesderived from pyrrole, furan, thiophene, imidazole, pyrazole, oxazole,isoxazole, thiazole, isothiazole, tetrazole,1,2,3,5-oxathiadiazole-2-oxide, triazolone, oxadiazolone, isoxazolone,oxadiazolidinedione, triazoles substituted by F, —CN, —CF₃ or—C(O)—O—(C₁-C₄)alkyl, 3-hydroxypyrro-2,4-dione, 5-oxo-1,2,4-thiadiazole,pyridine, pyrazine, pyrimidine, indole, isoindole, indazole,phthalazine, quinoline, isoquinoline, quinoxaline, quinazoline,cinnoline, carboline and benz-anellated, cyclopenta-, cyclohexa- orcyclohepta-anellated derivatives of said heterocycles are examples ofthe terms “—(C₅-C₁₄)-heterocycle”. The moieties 2- or 3-pyrrolyl,phenylpyrrolyl such as 4- or 5-phenyl-2-pyrrolyl, 2-furyl, 2-thienyl,4-imidazolyl, methylimidazolyl, e.g. 1-methyl-2-, -4- or -5-imidazolyl,1,3-thiazole-2-yl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-, 3- or4-pyridyl-N-oxide, 2-pyrazinyl, 2-, 4- or 5-pyrimidinyl, 2-, 3- or5-indolyl, substituted 2-indolyl, e.g. 1-methyl, 5-methyl, 5-methoxy-,5-benzyloxy-, 5-chloro- or 4,5-dimethyl-2-indolyl, 1-benzyl-2- or-3-indolyl, 4,5,6,7-tetrahydro-2-indolyl, cyclohepta[b]-5-pyrrolyl, 2-,3- or 4-quinolyl, 1-, 3- or 4-isoquinolyl,1-oxo-1,2-dihydro-3-isoquinolyl, 2-quinoxalinyl, 2-benzofuranyl,2-benzothienyl, 2-benzoxazolyl or benzothiazolyl or dihydropyridinyl,pyrrolidinyl, e.g. 2- or 3-(N-methylpyrrolidinyl), piperazinyl,morpholinyl, thiomorpholinyl, tetrahydrothienyl or benzodioxolanyl areparticularly preferred.

Preferred compounds of Formula I are, for example,ethyl-4-chloroacetoacetate, methylacetoacetate,ethyl-8-chloro-6-oxooctanoic acid, ethyl-3-oxovaleriate,4-hydroxy-2-butanone, ethyl-2-oxovaleriate, ethyl-2-oxo-4-phenylbutyricacid, ethyl pyruvate, ethyl phenyl glyoxylate, 1-phenyl-2-propanone,2-chloro-1-(3-chlorophenyl)ethane-1-one, acetophenone, 2-octanone,3-octanone, 2-butanone, 1-[3,5-bis(trifluoromethyl)phenyl]ethane-1-one,2,5-hexanedione, 1,4-dichloro-2-butanone, acetoxyacetone, phenacylchloride, ethyl-4-bromoacetoacetate, 1,1-dichloroacetone,1,1,3-trichloroacetone or 1-chloroacetone.

In the process according to the invention, the oxidoreductases can beused either in a completely purified or in a partially purified state orthe process can be performed with cells containing the oxidoreductasesaccording to the invention. In doing so, the cells used can be providedin a native, permeabilized or lysed state. The cloned oxidoreductasesaccording to SEQ ID No 1, SEQ ID No 2, SEQ ID No 3, SEQ ID No 4, SEQ IDNo 5, SEQ ID No 6, SEQ ID No 7, SEQ ID No 8, SEQ ID No 129 or homologuesthereof, respectively, are preferably used.

5.000 to 10 Mio U of oxidoreductase are used per kg of compound ofFormula I to be converted (no upper limit). The enzyme unit 1 Ucorresponds to the enzyme amount which is required for converting 1 μmolof the compound of Formula I per minute (min).

The enzymatic reduction itself proceeds under mild conditions so thatthe alcohols produced will not react further. The processes according tothe invention exhibit a high residence time and an enantiomeric purityof normally more than 95% of the chiral alcohols produced and a highyield, relative to the amount of keto compounds that is employed.

In the process according to the invention, the carbonyl compound is usedin an amount of from 3% to 50%, based on the total volume, preferablyfrom 5% to 40%, in particular from 10%-30%.

Furthermore, a preferred embodiment of the invention is characterized inthat the NAD or NADP formed during the reduction is continuously reducedto NADH or NADPH, respectively, with a cosubstrate.

In doing so, primary and secondary alcohols such as ethanol, 2-propanol,2-butanol, 2-pentanol, 3-pentanol, 4-methyl-2-pentanol, 2-heptanol,2-octanol or cyclohexanol are preferably used as the cosubstrate.

Said cosubstrates are reacted to the corresponding aldehydes or ketonesand NADH or NADPH, respectively, with the aid of an oxidoreductase andNAD or NADP, respectively. This results in a regeneration of the NADH orNADPH, respectively. The proportion of the cosubstrate for theregeneration thereby ranges from 5 to 95% by volume, based on the totalvolume.

For the regeneration of the cofactor, an additional alcoholdehydrogenase can be added. Suitable NADH-dependent alcoholdehydrogenases are obtainable, for example, from baker's yeast, fromCandida boidinii, Candida parapsilosis or Pichia capsulata. Furthermore,suitable NADPH-dependent alcohol dehydrogenases are present inLactobacillus brevis (DE 196 10 984 A1), Lactobacillus minor (DE 101 19274), Pseudomonas (U.S. Pat. No. 5,385,833) or in Thermoanaerobiumbrockii. Suitable cosubstrates for these alcohol dehydrogenases are thealready mentioned secondary alcohols such as ethanol, 2-propanol(isopropanol), 2-butanol, 2-pentanol, 4-methyl-2-pentanol, 2-octanol orcyclohexanol.

Furthermore, cofactor regeneration can also be effected, for example,using NAD- or NADP-dependent formate dehydrogenase (Tishkov et al., J.Biotechnol. Bioeng. [1999] 64, 187-193, Pilot-scale production andisolation of recombinant NAD and NADP specific Formate dehydrogenase).Suitable cosubstrates of formate dehydrogenase are, for example, saltsof formic acid such as ammonium formate, sodium formate or calciumformate. However, the processes according to the invention arepreferably carried out without such an additional dehydrogenase, i.e.,substrate-coupled coenzyme regeneration takes place.

The aqueous portion of the reaction mixture in which the enzymaticreduction proceeds preferably contains a buffer, e.g., a potassiumphosphate, tris/HCl or triethanolamine buffer, having a pH value of from5 to 10, preferably a pH value of from 6 to 9. In addition, the buffercan comprise ions for stabilizing or activating the enzymes, forexample, zinc ions or magnesium ions.

While carrying out the processes according to the invention, thetemperature is suitably in the range of from about 10° C. to 70° C.,preferably from 20° C. to 40° C.

In a further preferred embodiment of the processes according to theinvention, the enzymatic conversion is effected in the presence of anorganic solvent which is not miscible with water or miscible with wateronly to a limited extent. Said solvent is, for example, a symmetric orunsymmetric di(C₁-C₆)alkyl ether, a straight-chain or branched alkane orcycloalkane or a water-insoluble secondary alcohol that issimultaneously representing the cosubstrate. The preferred organicsolvents are, for example, diethyl ether, tertiary butyl methyl ether,diisopropyl ether, dibutyl ether, butyl acetate, heptane, hexane,2-octanol, 2-heptanol, 4-methyl-2-pentanol or cyclohexane. The solventcan, at the same time, also serve as a cosubstrate for cofactorregeneration.

If water-insoluble solvents and cosubstrates, respectively, are used,the reaction batch consists of an aqueous and an organic phase. Thesubstrate is distributed between the organic and aqueous phasesaccording to its solubility. The organic phase generally has aproportion of from 5 to 95%, preferably from 10 to 90%, based on thetotal reaction volume. The two liquid phases are preferably mixedmechanically so that a large surface is produced between them. Also inthis embodiment, the NAD or NADP, respectively, formed during theenzymatic reduction can be reduced back to NADH or NADPH, respectively,using a cosubstrate, as described above.

The concentration of the cofactor NADH or NADPH, respectively, in theaqueous phase generally ranges from 0.001 mM to 1 mM, in particular from0.01 mM to 0.1 mM.

In the processes according to the invention, a stabilizer of theoxidoreductase/dehydrogenase can, in addition, be used. Suitablestabilizers are, for example, glycerol, sorbitol, 1,4-DL-dithiothreitol(DTT) or dimethyl sulfoxide (DMSO).

The process according to the invention is carried out, for example, in aclosed reaction vessel made of glass or metal. For this purpose, thecomponents are transferred individually into the reaction vessel andstirred under an atmosphere of, e.g., nitrogen or air. The reaction timeranges from 1 hour to 48 hours, in particular from 2 hours to 24 hours.

Subsequently, the reaction mixture is processed. For this purpose, theaqueous phase is separated, the organic phase is filtered. The aqueousphase can optionally be extracted once more and can be processed furtherlike the organic phase. Thereupon, the solvent is optionally evaporatedfrom the filtered organic phase.

Furthermore, the invention relates to a process for obtaining chiralhydroxy compounds of Formula II,

R1-C(OH)—R2  (II)

wherein R¹ and R² are as defined above, which is characterized in that

-   -   a) a mixture containing the racemic compound of Formula II is        incubated in the presence of one of the oxidoreductases of the        invention according to SEQ ID No 1, SEQ ID No 2, SEQ ID No 3,        SEQ ID No 4, SEQ ID No 5, SEQ ID No 6, SEQ ID No 7, SEQ ID No 8,        SEQ ID No 129 or homologues thereof, NAD(P) and water, whereby        an enantiomer of the hydroxy compound of Formula II is converted        into the corresponding keto compound and NAD(P)H, and    -   b) the remaining enantiomer of the hydroxy compound of Formula        II is isolated.

If the carbonyl reductases according to SEQ ID No 1, SEQ ID No 3, SEQ IDNo 5, SEQ ID No 6, SEQ ID No 7, SEQ ID No 129 are used, thecorresponding chiral R-hydroxy compounds are preferably obtained. If thecarbonyl reductases according to SEQ ID No 2, SEQ ID No 4 and SEQ ID No8 are used, the corresponding chiral S-hydroxy compounds are preferablyobtained.

The reaction conditions are basically the same as in the above-mentionedprocess for the enantiospecific reduction of the keto compound ofFormula I. However, instead of an enantioselective reduction of the ketocompound of Formula I from the racemic mixture of the compound ofFormula II, only one enantiomer of the hydroxy compound of Formula II isoxidized enantioselectively into the corresponding keto compound. Thus,the opposite enantiomer of the hydroxy compound of Formula II remainsand can be isolated. Furthermore, instead of the alcohols used ascosubstrates, such as ethanol, 2-propanol (isopropanol), 2-butanol,2-pentanol or 2-octanol, the corresponding ketones thereof such asacetone are used in the process for the regeneration of the NAD. Forexample, the acetone and NAD(P)H are converted into NAD and isopropanolby means of the oxidoreductase according to the invention or anadditional dehydrogenase.

Preferred embodiments of the invention are illustrated in further detailby means of the following examples.

Example 1 Cultivation of Organisms and Screening for Carbonyl ReductaseActivity

For screening, the yeast strains Rhodotorula mucilaginosa DSMZ 70825,Pichia farinosa DSMZ 3316, Candida nemodendra DSMZ 70647, Pichiastipidis DSMZ 3651 and Pichia trehalophila DSMZ 70391, Lodderomyceselongisporus DSMZ 70320 were cultivated in the following medium: yeastextract (3), malt extract (3), peptone (5) and glucose (10) (the numbersin brackets are, in each case, g/l). The medium was sterilized at 121°C. and the yeasts were cultivated without further pH-adjustment at 25°C. and on a shaker at 160 revolutions per minute (rpm).

The strain Leuconostoc carnosum DSMZ 5576 was cultivated in thefollowing medium: glucose (20), yeast extract (5), meat extract (10),diammonium hydrogen citrate (2), sodium acetate (5), magnesium sulfate(0.2), manganese sulfate (0.05), dipotassium hydrogen phosphate (2). Themedium was sterilized at 121° C. and the organism was cultivated at 30°C. without further pH-adjustment or oxygen supply.

The strain Microbacterium spec. DSMZ 20028 was cultivated on a medium ofyeast extract (3) and trypticase soy flour (30) at 30° C. and with 160revolutions per minute (rpm).

The strain Gordonia rubripertincta DSMZ 43570 was cultivated on a mediumof yeast extract (4), glucose (4), malt extract (10) and CaCO₃ (2) at37° C. and with 160 revolutions per minute (rpm).

Subsequently, 125 mg of cells were resuspended with 800 μl of adigestion buffer (100 mM triethanolamine (TEA), pH=7.0), mixed with 1 gof glass beads and digested for 10 minutes (min) at 4° C. in the globemill (Retsch). The supernatant (lysate) obtained after 2 min ofcentrifugation at 12.000 rpm was used in the following activityscreening and for determining the enantiomeric excess (ee-value).Different ketones such as 2-butanone, 2-octanone,ethyl-4-chloroacetoacetate, acetophenone or ethyl-2-oxo-4-phenylbutyricacid were used as substrates.

Batch for activity screening:

860 μl 0.1 M KH₂PO₄/K₂PO₄ pH = 7.0 1 mM MgCl₂  20 μl NADPH or NADH (10mM)  20 μl lysate 100 μl substrate (100 mM)

The reaction was pursued for 1 min at 340 nm.

Batch for the determination of the ee-value:

20 μl lysate

100 μl NADH or NADPH (50 mM)

60 μl substrate (100 mM)

After 24 hours (h), the batches for ee-determination were extracted,e.g., with chloroform and the enantiomeric excess was determined via gaschromatography (GC). The enantiomeric excess is calculated as follows:

ee(%)=((R-alcohol−S-alcohol)/(R-alcohol+S-alcohol))×100.

TABLE 1 Activity in U/g cells host organism DSMZ 2-Butanone 2-OctanoneNo. Microorganism NADH NADPH NADH NADPH 70825 Rhodotorula <1 — <1 —mucilaginosa 3316 Pichia farinosa 12 — <1 — 70647 Candida 45 — 12 —nemodendra 3651 Pichia stipidis 10 — 6.4 — 70391 Pichia 85 — 45 —trehalophila 5576 Leuconostoc — 77 — 77 carnosum 20028 Microbacterium 9— 26 — spec. 43570 Gordonia 7.7 — 13 — rubripertincta 70320 Lodderomyces40 — 34 — elogisporus

DSMZ stands for Deutsche Sammlung für Mikroorganismen und Zellkulturen,Mascheroder Weg 1b, 38124 Braunschweig. Definition of enzyme units: 1 Ucorresponds to the enzyme amount which is required for converting 1 μmolof substrate per min.

Example 2 Isolation and Purification of NAD(P)H-Dependent MicrobialOxidoreductases

In order to isolate the NAD(P)H-dependent microbial oxidoreductases, themicroorganisms were cultivated as described under Example 1. Uponreaching the stationary phase, the cells were harvested and separatedfrom the medium by centrifugation. The enzyme release was effected bywet grinding using glass beads but may also be achieved by otherdigestion methods. For this purpose, for example, 100 g of wet cell masswere suspended with 400 ml of a digestion buffer (100 mMtriethanolamine, 1 mM MgCl₂, pH=7.0) and homogenized by means of aFrench press.

The crude extract obtained after centrifugation (7000 rpm) was thenpurified further via FPLC (fast protein liquid chromatography).

All oxidoreductases according to the invention could be purified bydifferent combinations of ion exchange chromatography, e.g., onQ-Sepharose Fast Flow (Pharmacia) or Uno Q (Biorad, Munich, Germany),hydrophobic interaction chromatography, e.g., on Octyl-Sepharose FastFlow or Butyl-Sepharose Fast Flow (Pharmacia), ceramic hydroxylapatitechromatography and gel permeation.

Example 2a Purification of an NADH-Dependent Oxidoreductase from Pichiafarinosa DSMZ 3316

For protein isolation, the lysate from Pichia farinosa DSMZ 3316obtained after centrifugation was directly applied to a Butyl-SepharoseFF-column equilibrated with 100 mM triethanolamine buffer pH=7.0 1 M(NH₄)₂SO₄ and was eluted with a decreasing linear salt gradient. Theoxidoreductase-containing fractions were combined and concentrated to anappropriate volume by means of ultrafiltration (exclusion limit 10 kDa).

Subsequently, the concentrated fractions of the oxidoreductase werefurther purified by Uno Q. For this purpose, the oxidoreductase wasdirectly applied to a UnoQ-column (Biorad) equilibrated with 50 mMpotassium phosphate buffer pH=7.0 and was eluted with an increasinglinear salt gradient, whereby the oxidoreductase eluted at 0 M NaClwithout binding whereas a major part of the impurities was bound andeluted at higher salt concentrations.

The third purification step was performed on a ceramic hydroxylapatitecolumn (Pharmacia), wherein the oxidoreductase was applied to a columnequilibrated with 10 mM potassium phosphate buffer, 1 mM MgCl₂ pH=6.8and was eluted with an increasing buffer concentration (400 mM potassiumphosphate buffer 1 mM MgCl₂ pH=6.8). The oxidoreductase was eluted at80-100 mM potassium phosphate buffer.

Thereupon, the molecular weight of the purified oxidoreductase obtainedwas determined via gel permeation (Superdex 200 HR; Pharmacia, 100 mMtriethanolamine, pH=7, 0.15 M NaCl). Catalase (232 kDa), aldolase (158kDa), albumin (69.8 kDa) and ovalbumin (49.4 kDa) were used as molecularweight standards.

The following Table 2 summarizes the results obtained.

TABLE 2 Total Activity activity [U/ml] [U] Specific activity Volume2-buta- 2-buta [U/mg] Purification step [ml] none none 2-butanone YieldCrude extract 70 1.3 80 0.1 100% Butyl-Sepharose 10 4.4 44 1.7 55% Uno Q1.4 17 24 5 30% Hydroxylapatite 0.7 13.5 9.5 16 12%

The enzyme activity of the oxidoreductase was determined in the testsystem according to Example 1 (batch activity screening), and thedetermination of the protein amount was performed according to Lowry etal. Journal of Biological Chemistry, 193 (1951): 265-275 or Peterson etal., Analytical Biochemistry, 100 (1979): 201-220). The quotient ofenzyme activity to protein amount yields the specific activity, whereinthe conversion of 1 μmol per min corresponds to 1 unit (U).

Example 2b Purification of an NADH-Dependent Oxidoreductase fromMicrobacterium spec. DSMZ 20028

For protein isolation, the lysate from Microbacterium spec. DSMZ 20028obtained after centrifugation was applied to a Q-Sepharose FF-columnequilibrated with 50 mM potassium phosphate buffer pH=7.0 and was elutedwith an increasing linear salt gradient. Thereby, the oxidoreductase waseluted at from 0.6 to 0.8 M NaCl. The oxidoreductase-containingfractions were combined and concentrated to an appropriate volume bymeans of ultrafiltration (exclusion limit 10 kDa).

Subsequently, the concentrated fractions of the oxidoreductase werefurther purified by Uno Q. For this purpose, the oxidoreductase wasdirectly applied to a UnoQ-column (Biorad) equilibrated with 50 mMpotassium phosphate buffer pH=7.0 and was eluted with an increasinglinear salt gradient, whereby the oxidoreductase eluted at 0.2-0.25 MNaCl.

The third purification step was performed on a ceramic hydroxylapatitecolumn (Pharmacia), wherein the oxidoreductase from Microbacterium spec.DSMZ 20028 was applied to a column equilibrated with 10 mM potassiumphosphate buffer, 1 mM MgCl₂ pH=6.8 and was eluted with an increasingbuffer concentration (400 mM potassium phosphate buffer 1 mM MgCl₂pH=6.8). The oxidoreductase was eluted at 80-100 mM potassium phosphatebuffer. Thereupon, the molecular weight of the purified oxidoreductaseobtained was determined as described under 2a.

The following Table 3 summarizes the results obtained.

TABLE 3 Total Activity activity [U/ml] [U] Specific activity Volume2-octa- 2-octa- [U/mg] Purification step [ml] none none 2-octanone YieldCrude extract 55 3.8 212 0.4 100% Q-Sepharose FF 34 4.1 139 0.56  65%Uno Q 0.8 9.3 7.5 3.8  3.5% Hydroxylapatite 0.5 4.2 2.1 117  1%

Example 3 Determination of the N-Terminal Sequence of an OxidoreductaseAccording to the Invention

After gel permeation in a 10% sodium dodecyl sulfate (SDS) gel, theenzyme preparations according to Example 2 were separated andtransferred onto a polyvinylidene difluoride membrane (PVDF-membrane).

The conspicuous band was subjected to N-terminal sequencing via Edmandegradation (Procise 492 (PE-Biosystems)).

Example 4 General Cloning Strategy of an Enantioselective AlcoholDehydrogenase Isolated from Yeasts

Chromosomal DNA is extracted according to the method described in“Molecular Cloning” by Manniatis & Sambrook. The resulting nucleic acidserves as a template for the polymerase chain reaction (PCR) withdegenerate primers. In doing so, 5′-primers are derived from the aminoacid sequence (SEQ ID No. 66; 72; 80) and 3′-primers are derived fromthe amino acid sequence (SEQ ID No. 67; 73, 81), involving the geneticcode specific for the organism (SEQ ID No. 68; 69; 74; 75; 82; 83).

Amplification is carried out in a PCR buffer [67 mM Tris-HCl (pH 8.3),16 mM (NH₄)₂SO₄, 115 mM MgCl₂, 0.01% Tween 20], 0.2 mM desoxynucleotidetriphosphate mix (dNTPs), 40 pMol of each primer and 2.5 U BioTherm StarPolymerase (Genecraft, Lüdingshausen, Germany)]. After activation of theBioTherm Star Polymerase (8 min 95° C.) and subsequent 45-50 cycles of aTouch-Down PCR, the reaction is cooled down to 4° C., and the entire PCRbatch is applied onto a 1% agarose gel for analysis.

The specific fragment resulting from the polymerase chain reaction isligated into the TA-cloning vecor pCR2.1 (Invitrogen, Karlsruhe,Germany) and sequenced with the primers M13 rev (SEQ ID NO 65) and M13uni (SEQ ID NO 128) with the aid of an ABI DNA sequencer.

The 5′- and 3′-terminal regions of the gene-coding sequence aredetermined using the RACE method (rapid amplification of cDNA ends).Based on the nucleic acid sequence of the specific fragment,oligonucleotides for 3′-RACE and 5′-RACE are constructed. Total RNAprepared from the cells serves as a template for the synthesis of thefirst cDNA strand using the 3′-RACE system (Invitrogen, Karlsruhe,Germany). This is followed by an amplification and a reamplification ofthe specific cDNA with the aid of 3′-RACE oligonucleotides (SEQ ID No.76; 77; 84; 85). Subsequently, the batch is applied onto a 1% agarosegel for analysis. The specific fragment carrying the missing 3′-flankingsequence information is isolated, ligated into a TA-cloning vectorpCR2.1 and sequenced.

The coding and non-coding 5′-terminal sequences are determined using the5′-RACE system (Invitrogen). For this purpose, mRNA from the total RNAobtained previously is enriched with the aid of Oligo dT-cellulose (NEB,Beverly, USA) and employed for the synthesis of the first cDNA strandwith the gene-specific oligonucleotides (SEQ ID No. 70; 71; 78; 79; 86;87). The subsequent amplification and reamplification of the specificcDNA results in a fragment which is ligated into a pCR2.1 TA-cloningvector (Invitrogen) for analysis. The plasmid containing the fragment isanalyzed with the aid of an ABI DNA sequencer. Thus, the missingsequence information about the 5′-end of the gene is obtained.

Rhodotorulla Protein mucilaginosa Pichia farinosa Pichia stipitisPartially VATAVETFGR LLTQTLALEQAK ADQVLLK sequenced (SEQIDNo 66)(SEQIDNo 72) (SEQIDNo 80) peptides FGEAVEQAR YNFTNKVAIITGGI ISFNLGDLALR(SEQIDNo 67) (SEQIDNo 73) (SEQIDNo 81) Primer for CCRAAYTCVACVGCVGTSGCYTGYTCYAANGCYAADGTYTG GCYGAYCARGTNTTRTTRAAR Touch-Down (SEQIDNO 68)(SEQIDNo 74) (SEQIDNo 82) PCR GCCTGYTCGACVGCYTCRCCCHAAYAARGTNGCHATHATYACHGG CTYAARGCYAARTCDCCYAAR (SEQIDNo 69) (SEQIDNo75) (SEQIDNo 83) Primer for CAACGTTCTGAAGAGATGACTTATGCTACCATGCCATGAGATTAG 3′-RACE (SEQIDNo 76) (SEQIDNo 84)GGTGGAGTGAAGTTATTGAC GCTGTAGACGTCGCTAAGAG (SEQIDNo 77) (SEQIDNo 85)Primer for CTCCGAGGTGTTGAGCGCATTG GCCATTCTTAGCCTGTTCGAGAGGATTCTCAAGGCTAAGTCAC 5′-RACE (SEQIDNo 70) (SEQIDNo 78) (SEQIDNo 86)GACGAGGTTCTTGATGTCGTCCTCC GTCATCTCTTCAGAACGTTGATCTT GATCTAACACCAGCTAATCT(SEQIDNo 71) (SEQIDNo 79) (SEQIDNo 87) CCAAAGGAGCTTATAGCAGTCT (SEQIDNo88)

Based on the sequence coding for the full-length gene (SEQ ID NO. 9; 10;11), specific primers for subsequent cloning of said DNA section into anappropriate expression system are constructed. For this purpose, forexample, 5′-primers with a recognition sequence for Nde I or with arecognition sequence for Sph I, or for BamHI, respectively, and3′-primers with a recognition sequence for Hind III are modified (SEQ IDNo. 89; 90; 91; 92; 93; 94; 95; 96).

In the subsequent PCR, chromosomal DNA serves as the template. The DNAsection coding for the respective oxidoreductase is amplified with theaid of Platinum pfx Polymerase (Invitrogen). After purification over 1%agarose gel, the resulting PCR product is treated with appropriate DNAendonucleases and ligated into the backbone of the pET21a vector(Novagen, Madison, USA) or into the backbone of the pQE70 vector(Qiagen, Hilden, Germany), respectively, which backbone has been treatedwith the same endonucleases.

After sequencing, the expression construct formed is taken into theexpression strain BL21 Star (Invitrogen) or RB791 (E. coli geneticstock, Yale, USA), respectively.

4a. Cloning of an Enantioselective Oxidoreductase from the Yeast Pichiafarinosa

For cloning the oxidoreductase from Pichia farinosa, chromosomal DNAwas, for example, extracted from the fresh cells of Pichia Farinosaaccording to the method described in “Molecular cloning” by Manniatis &Sambrook. The resulting nucleic acid served as a template for aTouch-Down PCR with oligonucleotides SEQ ID No. 74; 75. After 8 minutesof activating the Biotherm Star Polymerase in a PCR Cycler (BioRad,Hercules, USA), the following 30 temperature cycles were programmed foran identification of the specific DNA fragment:

94° C. 45 sec 60° C.-0.5° C./cycle 45 sec 68° C.  2 min

Subsequently, the amplification signal was increased by another 20cycles

94° C. 40 sec 52° C. 40 sec 72° C.  1 min.

After the fractionation of the entire reaction batch in 1% agarose gel,a specific fragment having a size of 550 bp was detected. Said fragmentwas eluted from the gel and ligated into the pCR2.1 TA-vector(Invitrogen, Karlsruhe, Germany). The plasmid pCR2.1-PF550 formed wassubjected to sequencing.

A sequence analysis of the gene fragment having a length of 550 bpshowed an open reading frame of 174 amino acid residues, in which thetwo sequence fragments of the N-terminus and of the internal peptidecould also be found.

Based on the nucleotide sequence of the fragment having a length of 521bp, oligonucleotides for a 3′-RACE (SEQ ID No 76; 77) and a 5′-RACE (SEQID No 78; 79; 88) were constructed. For the cDNA synthesis reaction, thetotal RNA from the cells of Pichia farinosa was prepared as follows.

600 mg of fresh cells were resuspended in 2.5 ml of ice-cold LETSbuffer. 5 ml (about 20 g) of glass beads washed in nitric acid andequilibrated with 3 ml phenol (pH 7.0) were added to said cellsuspension. The entire batch was then vortexed in each case for 30 sec,in total for 10 min, and was cooled on ice for 30 sec. Subsequently, 5ml of an ice-cold LETS buffer were added and thoroughly vortexed onceagain. Said cell suspension was centrifuged at 11000 g and at 4° C. for5 min. The aqueous phase was recovered and extracted twice with an equalvolume of phenol: chloroform: isoamyl alcohol (24:24:1). This wassubsequently followed by the extraction with chloroform. After the finalextraction, the total RNA was precipitated at −20° C. for 4 h by adding1/10 vol. of 5 M LiCl₂. The synthesis of the first cDNA strand wascarried out using the 3′RACE system (Invitrogen, Karlsruhe, Germany).Subsequently, the specific cDNA was amplified with the oligonucleotidesSEQ ID No 76 and AUAP (Invitrogen, Karlsruhe, Germany) in the reaction:67 mM Tris-HCl (pH 8.3), 16 mM (NH₄)₂SO₄, 115 mM MgCl₂, 0.01% Tween 20],0.2 mM desoxynucleotide triphosphate mix (dNTPs), 10 pMol of each primerand 2.5 U BioTherm Star Polymerase (Genecraft, Lüdingshausen, Germany)and with the following 30 temperature cycles: 94° C. 40 sec, 55° C. 40sec, 72° C. 1 min.

The PCR signal was increased via a nested PCR with primer SEQ ID No 77and primer UAP (Invitrogen, Karlsruhe, Germany) with 30 temperaturecycles: 94° C. 40 sec, 55° C. 40 sec, 72° C. 50 sec. The result was aspecific DNA fragment having a size of approximately 400 bp, which wasligated after isolation into the vector pCR2.1 (Invitrogen) from the 1%agarose gel. The sequence analysis of the DNA section having a length of382 bp yielded sequence information about the 3′-extension up to thestop codon and the poly-A loop of the cDNA coding for the oxidoreductasefrom Pichia farinosa.

For the 5′RACE reaction, 5 μg of total RNA prepared from the cells ofPichia farinosa were used. The synthesis of gene-specific cDNA wasperformed using the 5′RACE system (Invitrogen, Karlsruhe, Germany) andthe oligonucleotide SEQ ID No 78. The resulting gene-specific cDNA wassubjected to a homopolymeric dCTP addition reaction. This wassubsequently followed by an amplification of the cDNA in a PCR [67 mMTris-HCl (pH 8.3), 16 mM (NH₄)₂SO₄, 115 mM MgCl₂, 0.01% Tween 20], 0.2mM desoxynucleotide triphosphate mix (dNTPs), 20 pMol primer SEQ ID No79 and primer AAP (Invitrogen), 2.5 U BioTherm Star Polymerase(Genecraft, Lüdingshausen, Germany) and with the following 35temperature cycles: 94° C. 45 sec, 54° C. 45 sec, 72° C. 1 min 30 sec.The PCR signal was increased via a nested PCR with primer SEQ ID No 88and primer UAP (Invitrogen, Karlsruhe, Germany) with 30 temperaturecycles: 94° C. 40 sec, 55° C. 40 sec, 72° C. 1 min. The result was aspecific DNA fragment having a size of approximately 350 bp, which wasligated after elution into the vector pCR2.1 (Invitrogen) from the 1%agarose gel. The sequence analysis of the DNA segment having a length of352 bp yielded sequence information about the 5′-end of the cDNA codingfor the alcohol dehydrogenase/reductase.

Thus, the DNA segment coding for the protein has a total length of 765bp (SEQ ID No 10) and an open reading frame of 254 amino acids (SEQ IDNo 2). Chromosomal DNA of the Pichia farinosa cells was used as atemplate for the generation of the full-length DNA in a polymerase chainreaction [10 mM Tris-HCl, (pH 8.0); 50 mM KCl; 10 mM MgSO₄; 0.2 mM dNTPMix; 20 pMol Primer SEQ ID No 91 or, respectively, 20 pMol Primer SEQ IDNo 92, 20 pMol Primer SEQ ID No 93 and 2 U Platinum pfx Polymerase(Invitrogen)] and with temperature cycles:

Cycle 1 94° C., 2 min Cycle 2 × 30 94° C., 15 sec 56° C., 20 sec 68° C.,1 min 15 sec.

After purification over 1% agarose gel, the resulting PCR product wastreated with Nde I and Hind III, or with Sph I and Hind III,respectively, and was ligated into the backbone of the vector pET21a(Novagen, Madison, USA) or pQE70 (Qiagen, Hilden, Germany),respectively, which backbone had been treated with the sameendonucleases. After the transformation of 2 μl of the ligation batchinto E. coli Top10F′ cells, plasmid DNAs of ampicillin-resistantcolonies were checked for the correctness of the ligation that had beenperformed by means of a restriction analysis with the endonucleases NdeI or Sph I and Hind III, respectively. The DNA of the vectors positivefor the insert was transformed into the expression strain BL21 Star(Invitrogen) and RB791 (E. coli genetic Stock, Yale, USA), respectively.

Example 5 General Cloning Strategy of an Enantioselective OxidoreductaseIsolated from Bacteria

Genomic DNA is extracted according to the method described in “Molecularcloning” by Manniatis & Sambrook. The resulting nucleic acid serves as atemplate for the polymerase chain reaction (PCR) with degenerateprimers. In doing so, 5′-primers are derived from the amino acidsequence (SEQ ID No 104; 112) and 3′-primers are derived from the aminoacid sequence (SEQ ID No 105; 113), involving the genetic code specificfor the organism (SEQ ID No 106; 107; 114; 115).

Amplification is carried out in a PCR buffer [67 mM Tris-HCl (pH 8.3),16 mM (NH₄)₂SO₄, 115 mM MgCl₂, 0.01% Tween 20], 0.2 mM desoxynucleotidetriphosphate mix (dNTPs), 40 pMol of each primer and 2.5 U BioTherm StarPolymerase (Genecraft, Lüdingshausen, Germany)]. After activation of theBioTherm Star Polymerase (8 min 95° C.) and subsequent 45-50 cycles of aTouch-Down PCR, the reaction is cooled down to 4° C., and the entire PCRbatch is applied onto a 1% agarose gel for analysis.

The specific fragment resulting from the polymerase chain reaction isligated into the TA-cloning vecor pCR2.1 (Invitrogen, Karlsruhe,Germany) and sequenced with the primers M13 rev (SEQ ID No 65) and M13uni (SEQ ID No 128) with the aid of an ABI DNA sequencer.

The 5′- and 3′-terminal regions of the gene-coding sequence aredetermined using the inverse polymerase chain reaction method (iPCR).Based on the nucleic acid sequence of the specific internal fragment,oligonucleotides SEQ ID No 100; 101; 102; 103; 108; 109; 110; 111; 116;117; 118; 119 are constructed. Genomic DNA is digested by means of arestriction endonuclease and used in a religation so that smaller DNAsections can circulate. Said religation mixture is then used as atemplate for an iPCR and primers SEQ ID No 100; 102; 108; 110; 116; 118.The PCR signal is increased by a subsequent nested PCR with primers SEQID No 101; 103; 109; 111; 117; 119. The resulting specific fragment isligated after elution into the vector pCR2.1 (Invitrogen) from the 1%agarose gel.

Thus, the sequence analysis of the vector pCR2.1 containing the fragmentyields the missing sequence information about 3′- and 5′-coding regionsof the alcohol dehydrogenase/reductase gene.

Gordonia Protein Leuconostoc carnosum Microbacterium sp. rubropertinctaPartially NIEETTYEDWK MKALQYTKIGSHPE MKAIQIIQPG sequenced (SEQIDNo 97)(SEQIDNo 104) (SEQIDNo 112) peptides AYEALAAGTVV VGFFTQPYEVSVR (SEQIDNo105) (SEQIDNo 113) Primer for GACAGAWMGWTTNAARGGWAARGTHGCCTSCARTACACVAAGATCGG ATGAARGCNATYCARATYATYCARCC Touch-Down (SEQIDNo 98)(SEQIDNo 106) (SEQIDNo 114) PCR GCBGTRTAWCCNCCRTCDACDACRAAYTCGCBGCSAGBGCYTCRTABGC CYTCRTANGGYTGNGTRAARAA (SEQIDNo 99) (SEQIDNo 107)(SEQIDNo 115) Primer for CTAAGCCAATACCAAGTGTACCA TCCTCGCTGAGGCTCATCACGAGGACGAAGTCGTCCGAATG iPCR (SEQIDNo 100) (SEQIDNo 108) (SEQIDNo 116)GAACAAATCGTGCTACTGATTCATCAC GCTTCTCGATCTCGACGACTTC GCCGTCACCTTCAGCAACACC(SEQIDNo 101) (SEQIDNo 109) (SEQIDNo 117) GAAGAAGCCCAATCACAAAGAACTCGCGCAGCGAACTGATCGAG CTCGACGTGAGCGACGACAAG (SEQIDNo 102) (SEQIDNo 110)(SEQIDNo 118) GGCAGTCTATTTAGCTAGTGAAG GATCCAGCGCTACTCACTCGACGCAAGATCACCGGCAACGATG (SEQIDNo 103) (SEQIDNo 111) (SEQIDNo 119)

Based on the sequence coding for the full-length gene (SEQ ID No. 12;13; 14), specific primers for subsequent cloning of said DNA sectioninto an appropriate expression system are constructed. In doing so,5′-primers are modified with a recognition sequence for Nde I or with arecognition sequence for Sph I, or for BamHI, respectively, and3′-primers with a recognition sequence for Hind 111 (SEQ ID No. 120;121; 122; 123; 124; 125; 126; 127).

The amplification of the full-length DNA from genomic DNA, whichfull-length DNA codes for the protein, with subsequent restriction andligation into the expression vector is performed as described in Example3. The expression strain BL21 Star (Invitrogen) or RB791 (E. coligenetic stock, Yale, USA), respectively, is transformed with theexpression construct formed.

5a Cloning of an Enantioselective Alcohol Dehydrogenase/Reductase fromthe Microorganism Microbacterium sp.

For cloning the oxidoreductase from Microbacterium sp., genomic DNA was,for example, extracted from the fresh cells of Microbacterium sp.according to the method described in “Molecular cloning” by Manniatis &Sambrook. The resulting nucleic acid served as a template for a PCR with30 pMol each of oligonucleotides SEQ ID No. 106; 107. After 10 minutesof activating the Biotherm Star Polymerase in a PCR Cycler (BioRad,Hercules, USA), the following 30 temperature cycles were programmed foran identification of the specific DNA fragment:

94° C. 50 sec 60° C.  1 min 72° C.  1 min

After the fractionation of the entire reaction batch in 1% agarose gel,a specific fragment having a size of approximately 1000 bp was detected.Said fragment was eluted from the gel and ligated into the pCR2.1TA-vector (Invitrogen, Karlsruhe, Germany). The plasmid pCR2.1-Ms1000formed was subjected to sequencing.

A sequence analysis of the gene fragment having a length of 1002 bpshowed an open reading frame of 334 amino acid residues, in which thetwo sequence fragments of the N-terminus and of the internal peptidecould also be found.

Based on the nucleotide sequence of the fragment having a length of 1002bp, oligonucleotides (SEQ ID NO 108; 109; 110; 111) for an inverse PCR(iPCR) were constructed.

Genomic DNA (2.5 μg) from the cells of Microbacterium sp. was treated ina 50 μl batch with 20 U restriction endonuclease Sac I for 25 min. Afterthe phenol:chloroform:isoamyl:alcohol (25:24:1) extraction of the entirebatch and after precipitation with 1/10 vol. of 3M Na-acetate (pH5.2)and 2.5 vol. of ethanol, the DNA thus digested was transferred into 25μl H₂O. 5 μl (200 ng) thereof were used in a religation reaction in atotal volume of 40 μl and 2 U of T4 ligase (Fermentas). The religatedgenomic DNA (2 μl=20 ng) was then used in an iPCR [67 mM Tris-HCl (pH8.3), 16 mM (NH₄)₂SO₄, 115 mM MgCl₂, 0.01% Tween 20], 0.2 mMdesoxynucleotide triphosphate mix (dNTPs), 30 pMol of each primer (SEQID No 108; 110) with 2.5 U BioTherm Star Polymerase (Genecraft,Lüdingshausen, Germany)]. The amplification was conducted with thefollowing cycles:

Cycle 1 95° C., 10 min Cycle 2 × 30 95° C., 1 min 56° C., 1 min 72° C.,2 min

The amplification signal was increased in a nested PCR with theoligonucleotides SEQ ID No 109 and SEQ ID No 111.

Subsequently, the amplification reaction was cooled down to 4° C. andapplied as a whole onto a 1% agarose gel. The result was a specificfragment having a size of approximately 1000 bp. After the elution fromthe gel, the fragment was ligated into the pCR2.1 vector (Invitrogen,Karlsruhe, Germany).

The sequence analysis of the plasmid containing the fragment yieldedinformation about the 5′- and 3′-flanking sequences. Thus, the DNAsegment coding for the protein has a total length of 1044 bp ending in astop codon (SEQ ID No 13) and exhibits an open reading frame of 347amino acids (SEQ ID No 5).

Genomic DNA of Microbacterium sp. cells was used as a template for thegeneration of the full-length DNA coding for the protein in a polymerasechain reaction using the GC-Rich PCR system (Roche, Mannheim, Germany)and 30 pMol oligonucleotides SEQ ID No 123 or SEQ ID No 124,respectively, with 30 pMol oligonucleotide SEQ ID No 125 and temperaturecycles:

Cycle 1 95° C., 3 min Cycle 2 × 30 95° C., 30 sec 59° C., 30 sec 72° C.,45 sec.

After purification over 1% agarose gel, the resulting PCR product wastreated with Nde I and Hind III, or with Sph I and Hind III,respectively, and was ligated into the backbone of the vector pET21a(Novagen, Madison, USA) or pQE32 (Qiagen, Hilden, Germany),respectively, which backbone had been treated with the sameendonucleases. After the transformation of 2 μl of the ligation batchinto E. coli Top10F′ cells, plasmid DNAs of ampicillin-resistantcolonies were checked for the correctness of the ligation that had beenperformed by means of a restriction analysis with the endonucleases NdeI or Sph I and Hind III, respectively. The DNA of the vectors positivefor the insert was transformed into the expression strain BL21 Star(Invitrogen) and RB791 (E. coli genetic Stock, Yale, USA), respectively.

Example 6 Expression of Recombinant Alcohol Dehydrogenases/Reductases inE. coli

The Escherichia coli strains BL21 Star (Invitrogen, Karlsruhe, Germany)and RB791 (E. coli genteic stock, Yale, USA), respectively, which hadbeen transformed with the expression construct, were cultivated in 200ml of LB-medium (1% tryptone, 0.5% yeast extract, 1% NaCl) withampicillin (50 μg/ml) and carbenicillin (50 μg/ml), respectively, untilan optical density of 0.5 measured at 550 nm was achieved. Theexpression of recombinant protein was induced by the addition ofisopropyl thiogalactoside (IPTG) at a concentration of 0.1 mM. After 8hours or after 16 hours, respectively, of induction at 25° C. and 220rpm, the cells were harvested and frozen at −20° C. For the activitytest, 10 mg of cells were mixed with 500 μl of 100 mM TEA buffer pH 7.0and 500 μl of glass beads and were digested for 10 min using a globemill. The lysate obtained was then used for the respective measurementsin a diluted state. The activity test was composed as follows: 870 μl of100 mM TEA buffer pH 7.0, 160 μg NAD(P)H, 10 μl diluted cell lysate. Thereaction was started with the addition of 100 μl of a 100 mM substratesolution to the reaction mixture.

Expression Expression Activity vector strain Substrate U/g SEQ ID No 1pET21a BL21 Star acetophenone 4700 U/g SEQ ID No 2 pET21a BL21 Star2-butanone 1900 U/g SEQ ID No 3 pQE70 RB791 CLAEE 5220 U/g SEQ ID No 4pET21a BL21 Star CLAEE 8300 U/g SEQ ID No 5 pET21a BL21 Star 2-octanone8000 U/g SEQ ID No 6 pQE70 RB791 2-octanone 1600 U/g SEQ ID No 7 pET21aBL21 Star SEQ ID No 8 pET21a BL21 Star CLAEE 7000 U/g

Example 7 Characterization of the Recombinant Oxidoreductases

7a: pH-Optimum

The buffers listed in Table 4 were produced. The concentration of therespective buffer components in each case amounted to 50 mM.

TABLE 4 pH-value Buffer system 4 Na-acetate/acetic acid 4.5Na-acetate/acetic acid 5 Na-acetate/acetic acid 5.5 KH₂PO₄/K₂PO₄ 6KH₂PO₄/K₂PO₄ 6.5 KH₂PO₄/K₂PO₄ 7 KH₂PO₄/K₂PO₄ 7.5 KH₂PO₄/K₂PO₄ 8KH₂PO₄/K₂PO₄ 8.5 KH₂PO₄/K₂PO₄ 9 glycine/NaOH 9.5 glycine/NaOH 10glycine/NaOH 11 glycine/NaOH

Measuring batch (30° C.)-pH optimum reduction:

870 μl  of the buffer systems each mentioned in Table 3 20 μl of NAD(P)H10 mM 10 μl of a diluted enzyme

Incubation was performed for about 2 to 3 min, subsequently

100 μl of a substrate solution (100 mM) were added.

Depending on the oxidoreductase, 2-butanone or 2-octanone was used asthe substrate. The reaction was pursued for 1 min at 340 nm. In order todetermine the pH-optimum, the enzymatic reaction in the respectivebuffer listed in Table 4 was analyzed. In order to determine thepH-optimum for the oxidation reaction, NAD(P) was used as the cofactorand 2-propanol or 2-octanol was used as the substrate.

The results for the oxidoreductases according to the invention arecompiled in Table 5.

TABLE 5 DSMZ No. Microorganism pH-opt red pH-opt ox 70825 Rhodotorulamucilaginosa 7-8 8.0-9.5 3316 Pichia farinosa 5-6  7-11 70647 Candidanemodendra 6 10-11 3651 Pichia stipidis 5.5-6.5 6.5-7.5 70391 Pichiatrehalophila   7-7.5 7-8 5576 Leuconostoc carnosum 5.0-6   6.5-9.5 20028Microbacterium spec. 6.5-7.5 7.5-8.5 43570 Gordonia rubripertincta 57.5-9.5

7b: pH Stability

The determination of the activity of the recombinant oxidoreductases wasexamined by storing them in the buffer systems mentioned in Table 4. Forthis purpose, the different buffers (50 mM) were prepared in the rangeof from pH 4 to 11, and the oxidoreductase produced according to Example4 was diluted therewith. After 30, 60 and 120 minutes of incubation, 10μl were taken from the batch and used in the activity test according toExample 1.

The initial value is thereby the measured value which was obtainedimmediately after the dilution (1:20) of the enzyme in a potassiumphosphate buffer 50 mM pH=7.0. Under the given conditions, said valuecorresponded to an extinction change of approx. 0.70/min and was set asa 100% value, and all subsequent measured values were put in relation tothis value.

In Table 6, the pH ranges in which the enzymes exhibited no less than50% of the initial activity with an incubation lasting for 120 min arecompiled for the oxidoreductases according to the invention.

TABLE 6 DSMZ No. Microorganism pH-range stability 70825 Rhodotorulamucilaginosa 5.5-9.5 3316 Pichia farinosa  5.5-10.0 70647 Candidanemodendra 6.5-9.5 3651 Pichia stipidis 6.0-7.0 70391 Pichiatrehalophila 6.0-8.0 5576 Leuconostoc carnosum 4.5-9.5 20028Microbacterium spec. 5.0-9.5 43570 Gordonia rubripertincta 4.5-10 

7c: Temperature Optimum

In order to determine the optimum test temperature, the enzyme activityfor the oxidoreductases according to the invention was measured in thestandard measuring batch in a temperature range of from 15° C. to 70° C.

The temperature optima determined are compiled in Table 7:

TABLE 7 DSMZ No. Microorganism Topt 70825 Rhodotorula mucilaginosa 50°C. 3316 Pichia farinosa 40° C. 70647 Candida nemodendra 65° C. 3651Pichia stipidis 40° C. 70391 Pichia trehalophila n.b. 5576 Leuconostoccarnosum 60° C. 20028 Microbacterium spec. 60° C. 43570 Gordoniarubripertincta 45-55° C.  

7d: Temperature Stability

In an analogous manner as described under Example 5c, the temperaturestability was determined for the range of from 15° C. to 70° C. For thispurpose, a dilution of the oxidoreductases according to the inventionwas in each case incubated at the respective temperature for 60 min and180 min and was subsequently measured at 30° C. with the above-mentionedtest batch. In Table 8, the temperature ranges in which the enzymesexhibited no less than 50% of the initial activity with an incubationlasting for 120 min are compiled for the oxidoreductases according tothe invention.

TABLE 8 DSMZ No. Microorganism Temperature stability 70825 Rhodotorulamucilaginosa 15-35° C. 3316 Pichia farinosa 15-25° C. 70647 Candidanemodendra 15-35° C. 3651 Pichia stipidis 15-35° C. 70391 Pichiatrehalophila 15-35° C. 5576 Leuconostoc carnosum 15-35° C. 20028Microbacterium spec. 15-60° C. 43570 Gordonia rubripertincta 15-55° C.

7e: Substrate Spectrum

The substrate spectrum of the oxidoreductases according to the inventionwas determined by measuring the enzyme activity for reduction andoxidation with a number of ketones and alcohols. For this purpose, thestandard measuring batch according to Example 1 was used with differentsubstrates.

The activity with methyl acetoacetate was set to 100% for all enzymesand all the other substrates were put in relation thereto.

TABLE 9 Substrate spectra reduction Rhodotorula Pichia LeuconostocMicrobacterium Gordonia mucilaginosa farinosa Pichia stipidis carnosumspec. rubripertincta Substrate SEQ ID NO 1 SEQ ID NO 2 SEQ ID NO 3 SEQID NO 4 SEQ ID NO 5 SEQ ID NO 6 1-Phenyl-2- 66% 10% 30% 13% 80% 82%propanone Phenacyl 36% 130% 9% 37% <2% 7% chloride Acetophenone 12% 195%32% 28% 52% 23% Aceto- n.b. 25% 84% n.b. 125% 68% naphthone Butyro- 0%0% 0% 0% 0% 0% phenone 2-Octanone 71% 20% 27% 28% 227% 75% 3-Octanone29% 10% 40% 18% 47% 52% 2-Butanone 190% 65% 49% 36% 4% 14% Ethyl-2- 4%85% 60% 25% <2% 23% oxovaleriate Ethyl-2-oxo- 4% 35% 16% 10% <2% 18%4-phenyl butyric acid Ethyl 60% 560% 148% 122% 480% 160% pyruvate Ethylphenyl- 8% 35% 3% 4% <2% 11% glyoxylate Ethyl-4-chloro 79% 70% 100% 80%110% 110% acetoacetate Methyl 100% 100% 100% 100% 100% 100% acetoacetateEthyl-3- 60% 45% 73% 30% <2% 56% oxovaleriate Acetone 82% 55% 100% 28%<2% 7%

7f: Stability in the Aqueous/Organic Two-Phase System

The stability of the novel oxidoreductases in aqueous/organic two-phasesystems was examined by diluting the lysates obtained in Example 6 (froma recombinant expression) in an aqueous buffer suitable for therespective oxidoreductase (approx. 10 units/ml buffer). Then, the samevolume of an organic solvent not miscible with water was added to theoxidoreductase diluted in the buffer and the batch was incubated at roomtemperature with constant thorough mixing (thermomixer at 170 rpm).After 24 h of incubation, 10 μl each were taken from the aqueous phaseand used for the determination of the enzyme activity in the standardtest batch (potassium phosphate buffer (KPP) 100 mM, pH=7.0, 0.2 mMNAD(P)H, 10 mM substrate). Also in this case, the initial valueimmediately after the dilution in the buffer was set to 100%, and allfurther values were put in relation thereto.

TABLE 9 Enzyme activity after 24 h of incubation in the aqueous/organictwo-phase system Diiso- Butyl- Diethyl- propyl- Cyclo- System Bufferacetate ether MTBE ether Heptane hexane Rhodotorula 100% 80-100% 80-100%80-100% 80-100% 80-100% 80-100% mucilaginosa SEQ ID No 1 Pichia farinosa100% 40-60 60-80% 80-100% 40-60% 40-60 40-60% SEQ ID No 2 Candida 100%80-100% 80-100% 80-100% 80-100% 80-100% 80-100% nemodendra SEQ ID No 8Pichia stipidis 100% 40-60% n.b. 20-50% 60-80% 80-100% 40-60% SEQ ID No3 Leuconostoc 100% 80-100% 80-100% 80-100% 80-100% 80-100% 80-100%carnosum SEQ ID No 4 Microbacterium 100% 80-100% 80-100% 80-100% 80-100%80-100% 80-100% spec. SEQ ID No 5 Gordonia 100% 80-100% 80-100% 80-100%80-100% 80-100% 80-100% rubripertincta SEQ ID No 6 MTBE =Methyl-tert-butyl ether

TABLE 10 Substrate spectra oxidation Micro- Rhodotorula PichiaLeuconostoc bacterium Gordonia mucilaginosa farinosa Pichia stipidiscarnosum spec. rubripertincta Substrate SEQ ID NO1 SEQ ID NO2 SEQID NO3SEQID NO4 SEQID NO5 SEQID NO6 S-2-Octanol 100% 0% 100% 0% 100% 100%R-2-Octanol 0% 100% 0% 100% 0% 0% S-2-Butanol 266% 100% 237% 56% 5% 45%R-2-Butanol 60% 340% 74% 178% 0% 17% S-Phenyl-2- 200% 0% 26% 0% 10% 0%propanol R-Phenyl-2- 0% 0% 0% 6% 0% 0% propanol Ethyl-(S)-4- 0% 0% 0% 0%0% 0% chloro-3- hydroxy- butyrate Ethyl-(R)-4- 0% 0% 0% 0% 10% 0%chloro-3- hydroxy- butyrate 2-Propanol 180% 180% 218% 67% <1% 27%Cyclohexanol 26% 120% 0% n.b. n.b. 7%

Example 8 Preparative Conversions 8a: Synthesis ofmethyl-(3S)-3-hydroxypentanoate with oxidoreductase from Rhodotorulamucilaginosa

For the preparative batch, a mixture of 25 ml of a buffer (100 mM TEA,pH=7, 1 mM ZnCl₂, 10% glycerol), 375 ml 4-methyl-2-pentanol, 100 mlmethyl-3-oxopentanoate, 100 mg NAD and 37 kU recombinant oxidoreductasefrom Rhodotorula mucillaginosa DSMZ 70825 was incubated at roomtemperature for 24 h with constant thorough mixing. After 24 h, 97% ofthe methyl-3-oxopentanoate used had been reduced tomethyl-(3S)-3-hydroxypentanoate. Subsequently, the 4-methyl-2-pentanolphase containing the product was separated from the aqueous phase,filtered, and the product methyl-(3S)-3-hydroxypentanoate was obtainedby distillation.

In this manner, the product methyl-(3S)-3-hydroxypentanoate was obtainedin high yield with a purity of >99% and with an enantiomeric excess of>99.5%.

8b: Synthesis of (ZR)-1-chloropropane-2-ol with oxidoreductase fromPichia farinosa

For the conversion, a mixture of 80 ml of a buffer (100 mM TEA, pH=7, 1mM MgCl₂, 10% glycerol), 15 ml 2-propanol, 5 ml chloroacetone, 10 mg NADand 2 kU recombinant oxidoreductase from Pichia farinosa DSMZ 3316 wasincubated at room temperature for 24 h with constant thorough mixing.After 24 h, the chloroacetone used had been reduced completely to(2R)-1-chloropropane-2-ol. Subsequently, the reaction mixture wasextracted with ethyl acetate, the solvent was removed using a rotaryevaporator, and the crude product was obtained. The(2R)-1-chloropropane-2-ol produced in this manner has an enantiomericexcess of >99%.

8c: Synthesis of (R)-2-chloro-1-(3-chlorophenyl)ethane-1-ol withoxidoreductase from Pichia stipidis

For the conversion, a mixture of 20 ml of a buffer (100 mM potassiumphosphate, pH=8.5, 1 mM MgCl₂, 10% glycerol), 20 g2-chloro-1-(3-chlorophenyl)ethane-1-one dissolved in 80 ml4-methyl-2-pentanol, 10 mg NAD and 20 000 U recombinant oxidoreductasefrom Pichia stipidis DSMZ 3651 was incubated at room temperature for 24h with constant thorough mixing. After 24 h, more than 99% of the2-chloro-1-(3-chlorophenyl)ethane-1-one used had been reduced.Subsequently, the 4-methyl-2-pentanol phase containing the product wasseparated from the aqueous phase, filtered, and the product(R)-2-chloro-1-(3-chlorophenyl)ethane-1-ol was obtained by distillation.

In this manner, the product (R)-2-chloro-1-(3-chlorophenyl)ethane-1-olwas obtained in high yield with a purity of >98% and with anenantiomeric excess of >99.9%.

8d: Synthesis of ethyl-(S)-4-chloro-3-hydroxybutyric acid withoxidoreductase from Leuconostoc carnosum

For the conversion, a mixture of 8 mL of a buffer (100 mM TEA, pH=7, 1mM MgCl₂), 24 ml isopropanol, 8 ml ethyl-4-chloroacetoacetate, 2 mg NADPand 6.7 kU (=6 ml) recombinant oxidoreductase from Leuconostoc camosumDSMZ 5576 was incubated at room temperature for 24 h with constantthorough mixing. After 24 h, more than 99% of theethyl-4-chloroacetoacetate used had been reduced toethyl-(S)-4-chloro-3-hydroxybutyric acid. The reaction mixture wasreprocessed by first removing the 2-propanol using a rotary evaporator.Subsequently, the reaction mixture was extracted with ethyl acetate, thesolvent was removed using a rotary evaporator, and the crude product wasobtained. The crude product ethyl-(S)-4-chloro-3-hydroxybutyric acidobtained in this manner exhibited an enantiomeric excess of >99.5%.

8e: Synthesis of (1S)-1-[3,5-bis(trifluoromethyl)phenyl]ethane-1-ol withoxidoreductase from Microbacterium spec.

For the conversion, a mixture of 1 mL of a buffer (100 mM TEA, pH=7, 10%glycerol, 1 mM ZnCl₂), 3 ml 4-methyl-2-pentanol, 1 ml 1-[3,5bis-(trifluoro-methyl)phenyl]ethane-1-one, 2 mg NAD and 0.7 kUrecombinant oxidoreductase from Microbacterium spec. DSMZ 20028 wasincubated at room temperature for 24 h with constant thorough mixing.After 24 h, more than 90% of the 1-[3,5bis-(trifluoro-methyl)phenyl]ethane-1-one used had been reduced to(1S)-1-[3,5-bis(trifluoromethyl)phenyl]ethane-1-ol. Subsequently, the4-methyl-2-pentanol phase containing the product was separated from theaqueous phase, filtered, and the product(1S)-1-[3,5-bis(trifluoromethyl)phenyl]ethane-1-ol was obtained bydistillation. The crude product(1S)-1-[3,5-bis(trifluoromethyl)phenyl]ethane-1-one obtained in thismanner exhibited an enantiomeric excess of >99.5%.

1.-7. (canceled)
 8. A process for the enantioselective enzymaticreduction of a keto compound to the corresponding chiral hydroxycompound, the process comprising: reducing the keto compound with anoxidoreductase in the presence of a cofactor, characterized in that anoxidoreductase is used which: (a) is encoded by a nucleic acid sequencefrom the group of SEQ ID No 9, SEQ ID No 10, SEQ ID No 11, SEQ ID No 12,SEQ ID No 13 and SEQ ID No 14, SEQ ID No 15, SEQ ID No 16, SEQ ID No130; or (b) is encoded by a nucleic acid sequence the complementarystrand of which hybridizes with one of the nucleic acid sequencesmentioned in (a) under highly stringent conditions.
 9. A processaccording to claim 8, characterized in that the keto compound has thegeneral Formula I:R₁—C(O)—R₂  (I) wherein R₁ stands for one of the moieties: 1)—(C₁-C₂₀)-alkyl, wherein alkyl is linear-chain or branched; 2)—(C₂-C₂₀)-alkenyl, wherein alkenyl is linear-chain or branched andoptionally contains up to four double bonds; 3) —(C₂-C₂₀)-alkynyl,wherein alkynyl is linear-chain or branched and optionally contains upto four triple bonds; 4) —(C₆-C₁₄)-aryl; 5)—(C₁-C₈)-alkyl-(C₆-C₁₄)-aryl; 6) —(C₅-C₁₄)-heterocycle which isunsubstituted or substituted one, two or three times by —OH, halogen,—NO₂ and/or —NH₂; or 7) —(C₃-C₇)-cycloalkyl; wherein the moietiesmentioned above under 1) to 7) are unsubstituted or substituted one, twoor three times, independently of each other, by —OH, halogen, —NO₂and/or —NH₂; and R₂ stands for one of the moieties: 8) —(C₁-C₆)-alkyl,wherein alkyl is linear-chain or branched; 9) —(C₂-C₆)-alkenyl, whereinalkenyl is linear-chain or branched and optionally contains up to threedouble bonds; 10) —(C₂-C₆)-alkynyl, wherein alkynyl is linear-chain orbranched and optionally contains two triple bonds; or 11)—(C₁-C₁₀)-alkyl-C(O)—O—(C₁-C₆)-alkyl, wherein alkyl is linear orbranched and is unsubstituted or substituted one, two or three times by—OH, halogen, —NO₂ and/or —NH₂; wherein the moieties mentioned aboveunder 8) to 11) are unsubstituted or substituted one, two or threetimes, independently of each other, by —OH, halogen, —NO₂ and/or —NH₂.10. A cloning vector comprising: one or several nucleic acid sequencescoding for the oxidoreductase according to SEQ ID No:1, SEQ ID No:2, SEQID No:3, SEQ ID No:4, SEQ ID No:5, SEQ ID No:6, SEQ ID No:7, SEQ IDNo:8, SEQ ID No: 129 or homologues thereof.
 11. An expression vectorlocated in a bacterial, insect, plant or mammalian cell, the expressionvector comprising: a nucleic acid sequence which codes for theoxidoreductase according to SEQ ID No:1, SEQ ID No:2, SEQ ID No:3, SEQID No:4, SEQ ID No:5, SEQ ID No:6, SEQ ID No:7, SEQ ID No:8, SEQ IDNo:129 or homologues thereof and is linked to an expression controlsequence.
 12. A recombinant host cell which is a bacterial, insect,plant or mammalian cell and has been transformed or transfected with anexpression vector comprising: a nucleic acid sequence which codes forthe oxidoreductase according to SEQ ID No:1, SEQ ID No:2, SEQ ID No:3,SEQ ID No:4, SEQ ID No:5, SEQ ID No:6, SEQ ID No:7, SEQ ID No:8, SEQ IDNo:129 or homologues thereof and is linked to an expression controlsequence.
 13. An oxidoreductase for use in a process for theenantioselective enzymatic reduction of a keto compound to thecorresponding chiral hydroxy compound, said oxidoreductate beingcharacterized by the following: the oxidoreductase is encoded by anucleic acid sequence which is selected from the group consisting of SEQID No 9, SEQ ID No 10, SEQ ID No 11, SEQ ID No 12, SEQ ID No 13, SEQ IDNo 14, SEQ ID No 15, SEQ ID No 16, SEQ ID No 130, and by a nucleic acidsequence the complementary strand of which hybridizes with one of thesequences SEQ ID No 9, SEQ ID No 10, SEQ ID No 11, SEQ ID No 12, SEQ IDNo 13, SEQ ID No 14, SEQ ID No 15, SEQ ID No 16 or SEQ ID No 130 underhighly stringent conditions.
 14. An oxidoreductase as in claim 13,characterized in that the keto compound has the general Formula I:R₁—C(O)—R₂  (I) wherein R1 stands for one of the moieties: 1)—(C₁-C₂₀)-alkyl, wherein alkyl is linear-chain or branched; 2)—(C₂-C₂₀)-alkenyl, wherein alkenyl is linear-chain or branched andoptionally contains up to four double bonds; 3) —(C₂-C₂₀)-alkynyl,wherein alkynyl is linear-chain or branched and optionally contains upto four triple bonds; 4) —(C₆-C₁₄)-aryl; 5)—(C₁-C₈)-alkyl-(C₆-C₁₄)-aryl; 6) —(C₅-C₁₄)-heterocycle which isunsubstituted or substituted one, two or three times by —OH, halogen,—NO₂ and/or —NH₂; or 7) —(C₃-C₇)-cycloalkyl; wherein the moietiesmentioned above under 1) to 7) are unsubstituted or substituted one, twoor three times, independently of each other, by —OH, halogen, —NO₂and/or —NH₂; and R₂ stands for one of the moieties: 8) —(C₁-C₆)-alkyl,wherein alkyl is linear-chain or branched; 9) —(C₂-C₆)-alkenyl, whereinalkenyl is linear-chain or branched and optionally contains up to threedouble bonds; 10) —(C₂-C₆)-alkynyl, wherein alkynyl is linear-chain orbranched and optionally contains two triple bonds; or 11)—(C₁-C₁₀)-alkyl-C(O)—O—(C₁-C₆)-alkyl, wherein alkyl is linear orbranched and is unsubstituted or substituted one, two or three times by—OH, halogen, —NO₂ and/or —NH₂; wherein the moieties mentioned aboveunder 8) to 11) are unsubstituted or substituted one, two or threetimes, independently of each other, by —OH, halogen, —NO₂ and/or —NH₂.15. A process for the enantioselective enzymatic reduction of a ketocompound to the corresponding chiral hydroxy compound, wherein the ketocompound is reduced with an oxidoreductase in the presence of acofactor, characterized in that an oxidoreductase is used which has anamino acid sequence in which: (a) at least 70% of the amino acids areidentical to the amino acids of one of the amino acid sequences SEQ IDNo 1, SEQ ID No 6, and SEQ ID No 8; (b) at least 55% of the amino acidsare identical to the amino acids of the amino acid sequence SEQ ID No 2and which comprises one of the internal partial sequences EYKEAAFTN (SEQID No 27), NKVAIITGGISGIGLA (SEQ ID No 28), DVNLNGVFS (SEQ ID No 29),HYCASKGGV (SEQ ID No 30), NCINPGYI (SEQ ID No 31) or LHPMGRLGE (SEQ IDNo 32); (c) at least 65% of the amino acids are identical to the aminoacids of the amino acid sequence SEQ ID No 3 and which comprises one ofthe internal partial sequences CHSDLHAIY (SEQ ID No 35), GYQQYLLVE (SEQID No 36), TFDTCQKYV (SEQ ID No 37), LLTPYHAM (SEQ ID No 38), LVSKGKVKP(SEQ ID No 39), GAGGLGVNG (SEQ ID No 40), IQIAKAFGAT (SEQ ID No 41) orLGSFWGTS (SEQ ID No 42); (d) at least 75% of the amino acids areidentical to the amino acids of the amino acid sequence SEQ ID No 4; (e)at least 65% of the amino acids are identical to the amino acids of theamino acid sequence SEQ ID No 5; (f) at least 50% of the amino acids areidentical to the amino acids of the amino acid sequence SEQ ID No 7; or(g) at least 72% of the amino acids are identical to the amino acids ofthe amino acid sequence SEQ ID No 129.